Central respiratory chemoreception is a well known and exquisitely sensitive homeostatic mechanism by which the brain maintains physiologically appropriate levels of pH and PCO2 via regulation of breathing. Dysfunction of central chemoreception is implicated in various central disorders of breathing (e.g., sudden infant death, central congenital hypoventilation), underscoring the critical importance of this system. A fundamental understanding of this important sensory feedback system demands identification of the relevant sensors - both the neurons involved and their molecular detectors. In this regard, a discrete population of neurons in the retro-trapezoid nucleus (RTN) of the medulla oblongata fulfills key criteria expected for chemoreceptor neurons. However, key questions remain. It is not known if pH sensing is an intrinsic property of those neurons, as required for continued consideration as chemosensors, and the cellular/molecular basis for RTN neuronal pH sensitivity has not been elucidated. In order to address these fundamental issues, research proposed in this grant application applies sophisticated molecular and in vitro electrophysiological techniques in the context of a new line of mice that express green fluorescent protein (GFP) selectively in chemosensitive RTN neurons. The hypothesis underpinning Specific Aim 1 is that RTN neurons are intrinsically chemosensitive, and that characteristic pH responses for different types of RTN neurons reflect distinct complements of background channels. In one sub-aim, experiments provide detailed characterization of pH-sensitive background K+ current and TTX-resistant leak Na+ current in RTN neurons recorded in brainstem slices, and they test if the leak Na+ current is carried by NALCN channels by using lentiviral-mediated shRNA knockdown. In a second sub-aim, GFP-expressing RTN neurons are recorded in a dissociated cell system to test definitively if pH sensitivity is an intrinsic property of RTN neurons. The hypothesis driving Specific Aim 2 is that the K+ channel(s) responsible for RTN neuronal chemosensitivity have significant constitutive activity at resting membrane potentials and they are intrinsically sensitive to pH or they are downstream effectors for pH-activated G protein-coupled receptors. Three sub-aims test involvement of different candidate pH-sensors that are expressed in RTN neurons; these include distinct K+ channels of the voltage-gated (KV) and two-pore-domain (K2P) channel family, as well as proton-activated G protein-coupled receptors. For this aim, recordings are obtained in GFP-expressing RTN neurons after functional expression of candidate molecular sensors is disrupted by genetic knockout or lentiviral-mediated expression of dominant-negative or shRNA constructs. The proposed studies provide critical information regarding this important homeostatic regulatory system. Identification of novel molecular substrates that underlie central respiratory chemoreception could provide new targets for therapeutic intervention in disorders of breathing.